There are basically two approaches which have successfully been used to transform plants genetically, but each method has limitations when applied in particular to monocotyledonous plants (monocots) and particularly to the commercially important crops, the cereals.
The first method utilizes Agrobacterium tumefaciens, which is a soil microbe containing a Ti plasmid that transfers DNA from the plasmid to the genome of the infected plant. The method is basically restricted to dicotyledonous plants since monocots are usually not susceptible to Agrobacterium. Although the genetic transformation of Asparagus using this method has been disclosed [WO 86/03776] and Grimsley et al., Nature 325:177-179 (1987), have reported the transfer of DNA into cereals via Agrobacterium, it had at the time of this invention not yet been demonstrated that actual transformation i.e., uptake and integration of the exogenous DNA with the host cell genome or expression of the desired new traits, may occur.
The second transformation method involves direct transfer of DNA into plant protoplasts. Such direct transfer can be obtained, for example, by chemically stimulated uptake using polyethylene glycol [Paszkowski et al., Meth. Enzym. 118:668-684 (1986)] by a high-voltage pulse (electroporation) which punches transient holes in the protoplast membrane. (See, e.g., Fromm et al., Nature 319:791-793 (1986)) or by particle bombardment (Sanford et al. Part. Sci. Technol 5(1) 27-37 (1987). The direct transfer method depends on a tissue culture system that allows regeneration of mature, fertile plants from protoplasts. However, in the cereals, it had at the time of this invention been possible to regenerate only rice from protoplasts. Attempts to regenerate maize protoplasts into fertile plants had been unsuccessful. See, e.g., Graves et al., Theor. Appl. Genet. 54:209-214 (1979).
It has recently been demonstrated that naked RNA can be introduced and expressed in whole cells of dicotyledonous plants (dicots) by means of electroporation. Morikawa et al., Gene 41:121-124 (1986), have shown that tobacco mosaic virus (TMV) RNA may be introduced into mesophyll cells of Nicottana, through intact cell walls. This is the first and only presently known example of introduction of naked genetic material into intact cells and is stated to have produced cells with a low survival rate. It was not clearly demonstrated that the cells were actually transformed rather than infected. Additionally, the procedure has been shown to work only on individual cells of dicotyledonous plants. Even if electroporation of individual monocot cells was to be successful in the future, there is still the unsolved problem of regenerating monocot, and in particular maize cells into whole fertile plants.